| Bone is a tissue characteristic of highly active metabolism.Bone homeostasis is maintained in a constantly dynamic balance between osteoblast-led bone formation and osteoclast-led bone resorption,and any disregulation from either side could cause diseases including osteoporosis,renal osteodystrophy,endocrine bone disease and osteogenesis imperfecta(OI).Skeletal diseases can cause bone pain,fractures,skeletal deformities,etc.,which could seriously affect patient's health and daily life.Drugs that regulate bone homeostasis mainly consist of bone resorption inhibitors and bone formation promoters,of which bone resorption inhibitors are clinically preferred.Recombinant human parathyroid hormone is the only bone formation promoter available today,but its clinical application has been limited due to its carcinogenic risk.Recently,extracts derived from natural plants and fruits,such as Chinese traditional medicine,have been widely used in treatments for bone diseases due totheir various advantages including sufficient resource,definite curative effect and minimal adverse reactions.Therefore,in this study,we selected curcumin and Solanum muricatum Ait(SM)extracts to explore their effects on osteogenic differentiation of mesenchymal stem cells,and their effects on OI,as well as the underlying mechanisms.PART I ObjectiveWe aimed to investigate the effects of curcumin and SM on osteogenic differentiation of adipose-derived mesenchymal stem cells(a MSCs)and bone marrow mesenchymal stem cells(BMSCs),respectively.We also attempted to explore whether the wingless gene-related integration site(Wnt)/?-catenin signal pathway was responsible for the observed effects.Methods1 Effect of curcumin on hydrogen peroxide-induced differentiation of a MSCs and Wnt/?-catenin signaling pathwayHuman adipose tissue was collected,isolated and induced to differentiate in osteogenic differentiation medium.Surface markers of a MSCs were determined by flow cytometry.Cells were treated with different concentrations of hydrogen peroxide and curcumin,followed by evaluations of cell viability,glutathione(GSH)levels,superoxide dismutase(SOD)activity,malondialdehyde(MDA)content and alkaline phosphatase(ALP)activity,as well as calcium content by alizarin red sulfate(ARS)staining assay.m RNA expression levels of Osteopontin(OPN),Collagen 1(Col 1),Osteoprotegerin(OPG)and Cyclin D1 were determined by RT-PCR.After a MSCs were treated with Wnt inhibitor Dickkopf1(DKK1),protein expression levels of?-catenin,Cyclin D1,Glycogen Synthase Kinase-3?(GSK-3?),p-GSK-3? and C-myc were determined by Western blot.2 Effect of SM on differentiation of BMSCs and Wnt/?-catenin signaling pathwaySM was prepared by phytochemistry followed by identification of its extracts.BMSCs were treated with osteogenic differentiation medium and SM extract,and the cytotoxicity of SM extract was evaluated by measuring cell viability and release of lactate dehydrogenase(LDH).BMSCs were cultured in osteogenic differentiation medium for 12 and 20 days for ALP and ARS staining assays,respectively.Bone Morphogenetic Protein 4(BMP4),Runt-related Transcription Factor 2(Runx2),?-catenin and Cyclin D1 m RNA and protein expression levels were determined by RT-PCR and Western Blot,respectively.Results1 Effect of Curcumin on Osteogenic Differentiation of a MSCs1.1 The obtained a MSCs were positive for CD29,CD44 and CD105 and negative for CD34 and CD45 cell surface markers.1.2 Compared with the control group,0-2 mmol/L hydrogen peroxide decreased cell viability of a MSCs in a dose-dependent manner.On the other hand,50 and 100mmol/L curcumin significantly decreased cell viability of a MSCs.Of note,0-20mmol/L curcumin prevented hydrogen peroxide-induced decrease in a MSC viabilityin a dose-dependent manner,while 50 and 100 mmol/L curcumin synergized with hydrogen peroxide to cause further reduction in cell viability.1.3 Compared with the control group,0.2-2 mmol/L hydrogen peroxide and50,100 ?mol/L curcumin significantly decreased,whereas 5-20 ?mol/L curcumin significantly increased ALP activity and calcium content in a MSCs,with statistically significant differences(P<0.05).Compared with the hydrogen peroxide group,ALP activity and calcium content in the hydrogen peroxide+20 ?mol/L curcumin group significantly increased(P<0.05).1.4 Compared with the control group,0.2 mmol/L hydrogen peroxide significantly decreased OPN and Col 1 while increased RANKL m RNA expression levels in a MSCs(P<0.05);20 ?mol/L curcumin significantly increased OPN and Col1 m RNA expressions in a MSCs(P<0.05).Compared with the hydrogen peroxide group,the expression levels of OPN and Col 1 m RNA were significantly decreased,whereas RANKL m RNA was significantly increased(P<0.05).1.5 Compared with the control group,0.2 mmol/L hydrogen peroxide significantly decreased GSH and SOD activity and increased MDA content in a MSCs(P<0.05);whereas 20 ?mol/L curcumin significantly increased GSH and SOD activity and decreased MDA content in a MSCs(P<0.05).Compared with the hydrogen peroxide group,GSH level and SOD activity were significantly increased and MDA content was significantly decreased in the hydrogen peroxide+20 ?mol/L curcumin group(P<0.05).1.6 Compared with the control group,0.2 mmol/L hydrogen peroxide significantly decreased protein expressions of ?-catenin,C-myc and Cyclin D1 andincreased protein expressions of p-GSK-3? and GSK-3? in a MSCs(P<0.05);whereas20 ?mol/L curcumin significantly increased protein expression of ?-catenin and decreased protein expressions of p-GSK-3? and GSK-3? in a MSCs(P<0.05).Compared with the hydrogen peroxide group,protein expressions of ?-catenin,C-myc and Cyclin D1 were significantly increased,and protein expressions of p-GSK-3? and GSK-3? were significantly decreased in the hydrogen peroxide+20?mol/L curcumin group(P<0.05).1.7 Compared with the control group,Wnt3 a and Wnt3a+curcumin up-regulated?-catenin,C-myc and Cyclin D1 protein expressions and down-regulated p-GSK-3?and GSK-3? protein expressions(P<0.05),but the effects of these two interventions were not statistically significant(P>0.05);DKK1 and DKK1+curcumin significantly down-regulated ?-catenin,C-myc and Cyclin D1 protein expressions and up-regulated p-GSK-3? protein expression(P<0.05).Compared with the DKK1 group,the protein expressions of C-myc and Cyclin D1 were significantly up-regulated in DKK1+curcumin group(P<0.05).1.8 Compared with the hydrogen peroxide group,ALP activity and calcium content were significantly increased in hydrogen peroxide+Wnt3a and hydrogen peroxide+Wnt3a+curcumin groups(P<0.05);compared with the hydrogen peroxide+curcumin group,ALP activity and calcium content were significantly decreased in hydrogen peroxide+curcumin+DKK1 group(P<0.05).2 Effects of SM Extract on Osteogenic Differentiation of BMSCs2.1 The main components of SM extract were total phenolic acid and total flavonoids,which were 1355±171 mg/100 g and 931±108 mg/100 g,respectively.2.2 Treating BMSCs with 50-500 ?g/m L SM extract for 1 d did not cause significant changes in cell viability and LDH release,but treating BMSCs with 500?g/m L SM extract for 3 d and 5 d significantly decreased cell viability and increased LDH release(P<0.05).2.3 Compared with the control group,the positive staining rate,mineralized nodule number,ALP activity,OPN and Col 1 m RNA expressions were all significantly increased in the middle and high dose SM extract groups(P<0.05).2.4 Compared with the control group,the m RNA expressions of Runx2 and BMP4 were significantly increased in the middle and high dose SM extract groups,and the m RNA expressions of ?-catenin and Cyclin D1 were significantly increased in the high dose SM extract group(P<0.05).2.5 Compared with the control group,the ALP activity and the number of mineralized nodules were significantly increased in the SM extract group;compared with the SM extract group,the ALP activity and the number of mineralized nodules were significantly reduced in the SM extract+DKK1 and the SM extract+noggin groups,with statistically significant differences(P<0.05).2.6 Compared with the control group,the m RNA and protein expressions of Runx2,BMP4,?-catenin and Cyclin D1 were all significantly increased in the SM extract group;compared with the SM extract group,the m RNA expressions of?-catenin and Cyclin D1 were significantly decreased in the SM extract+DKK1 group,while the m RNA expressions of Runx2 and BMP4 were significantly decreased in SM extract+noggin group,with statistically significant differences(P<0.05).Conclusions1 Low-dose curcumin can attenuate the hydrogen peroxide-induced oxidative stress in MSCs by anti-oxidation and activation of Wnt/?-catenin signaling pathway,thereby promoting cell osteogenic differentiation.2 SM extract is rich in flavonoids and phenolic compounds,which can promote osteogenic differentiation of BMSCs by activating the Wnt and BMP signaling pathways.PART II ObjectiveTo study the effect of SM extract on bone of OI mice and analyze the effect on bone Collagen 1n order to further discove the osteogenic properties of SM extract.MethodsThe OI model of mice was established.Micro CT was used to collect and analysis bone mass and microarchitectural parameters at vertebrae and femur metaphysis in the OI mice.Raman spectrometry was applied to identify change of bone mineral and matrix composition after SM treatment.Collagen synthesis marker N-Terminal Propeptide of Type ? Procollagen(PINP)and collagen degradationmarker Collagen Carboxyl Terminal Peptide(CTX)were detected by using enzyme immunoassay.ResultsSM extract could improve the microarchitectural parameters both at vertebrae and at femur metaphysis such as the bone volume fraction(Bone Volume over Total Volume,BV/TV),trabecular thickness(Trabecular Thickness,Tb Th),trabecular thickness(Trabecular Thickness,Tb Th)and bone junction density(Connectivity Density,Conn D).It also significantly increased the PO4/CH2,PO4/amide I and hydroxyproline content.SM extract could up-regulate PINP and down-regulate CTX expression.ConclusionSM extract can significantly ameliorate the symptoms in the OI mice,holding potential for developing new drugs of bone formation and bone remodeling. |
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